The interaction of polarized epithelial cells with the external environment is determined by their expression and presentation of plasma membrane proteins at their apical surfaces. The array of ion pumps, ion channels and receptors is regulated to a great extent by the trafficking and recycling of these protein constituents specifically to the apical pole. Much of the specificity of the recycling process is regulated through the Rab11a-containing apical recycling system. Recycling is regulated by a family of Rab11 interacting proteins (Rab11-FIPs) that are targeted to recycling vesicles based on their interaction with Rab11a. In addition, movement of cargoes through and out of the recycling system back to the apical membrane requires the molecular motor myosin Vb, which also interacts with Rab11a. We have hypothesized that multi-protein complexes involving Rab11a and its effectors regulate the trafficking of cargoes through the apical recycling system. Because of the complexity of the trafficking system, the proteins associated with recycling system machinery as well as the cargoes passing through this dynamic organelle are not readily predictable. We have previously prepared highly purified populations of recycling vesicles from human parietal cells by immunoisolation and begun a proteomic examination of tubulovesicle-associated proteins. To address the spectrum of trafficking regulation through the recycling system, we will pursue two specific aims: First, we will identify novel components of immunoisolated recycling vesicle populations prepared from human gastric parietal cells and will determine their roles in regulating recycling in epithelial cells. Second we will identify the components of putative multiprotein myosin Vb motor complexes associated with recycling vesicles and determine the structural basis of interactions among complex constituents. These studies should provide general insights into the functional relevance of components of the apical recycling system, including cargoes, regulatory protein complexes and signaling systems.